Bottom Line:
The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions.The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation.The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

ABSTRACTNosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

f6: The enzymatic assay performed on HPLC systems:(A) from top to bottom, only substrate as a control, NosA1-120, NosA1-140 variants, full-length NosA, and the catalytic reaction product nosiheptide used as another control; (B) from top to bottom, only substrate as a control, NosA1-111 plus Did2, NosA1-111 plus JmjN from KDM5C, NosA1-111 plus Vps60, and the catalytic reaction product nosiheptide used as another control. In (A,B), the controls substrate and the product nosiheptide were indicated by pink and green dotted line, respectively.

Mentions:
To probe whether the whole sequence of NosA112-151 affects the catalytic reaction, we measured the catalytic activities of the truncated NosA variants with different length, and found that the NosA1-140 variant maintained the catalytic activity almost similar to the full-length NosA, whereas NosA1-120 significantly lost the catalytic activity (Fig. 6A), revealing that the residues from 120 to 140 (i.e., A121FDPASPEPLTRPQEFVPPG140) of NosA112-151 are important to the catalytic reaction. To investigate whether the sequence and the coiled-coil conformation of NosA112-151 are specific to the catalytic reaction, we replaced NosA112-151 in the mixture by three randomly-selected peptides (Did2, Vps60 and JmjN) available in the lab19202122, respectively, and tested the catalytic activities of these mixtures, all displaying no catalytic activities at all (Fig. 6B). These observations suggest that the sequence and the flexible loop conformation of the NosA112-151 variant is crucial to the catalytic reaction.

f6: The enzymatic assay performed on HPLC systems:(A) from top to bottom, only substrate as a control, NosA1-120, NosA1-140 variants, full-length NosA, and the catalytic reaction product nosiheptide used as another control; (B) from top to bottom, only substrate as a control, NosA1-111 plus Did2, NosA1-111 plus JmjN from KDM5C, NosA1-111 plus Vps60, and the catalytic reaction product nosiheptide used as another control. In (A,B), the controls substrate and the product nosiheptide were indicated by pink and green dotted line, respectively.

Mentions:
To probe whether the whole sequence of NosA112-151 affects the catalytic reaction, we measured the catalytic activities of the truncated NosA variants with different length, and found that the NosA1-140 variant maintained the catalytic activity almost similar to the full-length NosA, whereas NosA1-120 significantly lost the catalytic activity (Fig. 6A), revealing that the residues from 120 to 140 (i.e., A121FDPASPEPLTRPQEFVPPG140) of NosA112-151 are important to the catalytic reaction. To investigate whether the sequence and the coiled-coil conformation of NosA112-151 are specific to the catalytic reaction, we replaced NosA112-151 in the mixture by three randomly-selected peptides (Did2, Vps60 and JmjN) available in the lab19202122, respectively, and tested the catalytic activities of these mixtures, all displaying no catalytic activities at all (Fig. 6B). These observations suggest that the sequence and the flexible loop conformation of the NosA112-151 variant is crucial to the catalytic reaction.

Bottom Line:
The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions.The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation.The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.

ABSTRACTNosiheptide is a parent compound of thiopeptide family that exhibit potent activities against various bacterial pathogens. Its C-terminal amide formation is catalyzed by NosA, which is an unusual strategy for maturating certain thiopeptides by processing their precursor peptides featuring a serine extension. We here report the crystal structure of truncated NosA1-111 variant, revealing three key elements, including basic lysine 49 (K49), acidic glutamic acid 101 (E101) and flexible C-terminal loop NosA112-151, are crucial to the catalytic terminal amide formation in nosiheptide biosynthesis. The side-chain of residue K49 and the C-terminal loop fasten the substrate through hydrogen bonds and hydrophobic interactions. The side-chain of residue E101 enhances nucleophilic attack of H2O to the methyl imine intermediate, leading to Cα-N bond cleavage and nosiheptide maturation. The sequence alignment of NosA and its homologs NocA, PbtH, TpdK and BerI, and the enzymatic assay suggest that the mechanistic studies on NosA present an intriguing paradigm about how NosA family members function during thiopeptide biosynthesis.